4-dedimethylaminotetracycline derivatives

ABSTRACT

The present invention provides new chemically modified 4-dedimethylaminotetracycline compounds that can be substituted with aryl, alkenyl, or alkynyl groups. The 7, 8, and/or 9 positions and methods for preparing such compounds. Other tetracycline compounds include the 4-dedimethylaminotetracycline derivatives with an oxime group, NH-Alkyl, or N—NH-Alkyl group substituted at the C4 position as well as C2 Mannich derivatives. The present invention also provides a method of treating a mammal suffering from conditions or diseases by administering to the mammal an effective amount of the new chemically modified tetracycline compounds.

The present application is a continuation of Ser. No. 10/458,076, filedJun. 10, 2003, now U.S. Pat. No. 6,894,036, which is a divisional ofSer. No. 09/911,861, filed on Jul. 24, 2001, Now U.S. Pat. No.6,638,922, which is a continuation-in-part of Ser. No. 09/573,654, filedMay 18, 2000, now U.S. Pat. No. 6,506,740, which is acontinuation-in-part of Ser. No. 09/479,604, filed Jan. 7, 2000, nowabandoned, which is a continuation-in-part of Ser. No. 09/195,013 filedNov. 18, 1998, now abandoned, all of which are incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to novel 4-dedimethylaminotetracyclinederivatives, methods for producing the novel derivatives and methods ofusing these derivatives.

BACKGROUND OF THE INVENTION

The compound, tetracycline, exhibits the following general structure:

The numbering system of the ring nucleus is as follows:

Tetracycline as well as the 5-OH (Terramycin) and 7-Cl (Aureomycin)derivatives exist in nature, and are well known antibiotics. Naturaltetracyclines may be modified without losing their antibioticproperties, although certain elements of the structure must be retained.The modifications that may and may not be made to the basic tetracyclinestructure have been reviewed by Mitscher in The Chemistry ofTetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978).According to Mitscher, the substituents at positions 5-9 of thetetracycline ring system may be modified without the complete loss ofantibiotic properties. Changes to the basic ring system or replacementof the substituents at positions 1-4 and 10-12, however, generally leadto synthetic tetracyclines with substantially less or effectively noantimicrobial activity. Some examples of chemically modifiednon-antimicrobial tetracyclines (hereinafter CMT) include4-dedimethylaminotetracyline,4-dedimethylaminosancycline(6-demethyl-6-deoxy-4-dedimethylaminotetracycline),4-dedimethylaminominocycline(7-dimethylamino-4-dedimethylaminotetracycline),and4-dedimethylaminodoxycycline(5-hydroxy-6-deoxy-4-dedimethyaminotetracycline).

Some 4-dedimethylaminotetracyline derivatives are disclosed in U.S. Pat.Nos. 3,029,284 and 5,122,519. They include6-demethyl-6-deoxy-4-dedimethylaminotetracycline and5-hydroxy-6-deoxy-4-dedimethylaminotetracycline with hydrogen and othersubstituents at the C7, and the C9 positions on the D ring. Thesesubstituents include amino, nitro, di(lower alkyl)amino, and mono(loweralkyl)amino or halogen. The6-demethyl-6-deoxy-4-dedimethylaminotetracycline derivatives and5-hydroxy-6-deoxy-4-dedimethylaminotetracycline derivatives are said tobe useful as antimicrobial agents.

Other 4-dedimethylaminotetracycline derivatives with an oxime group atthe C4 position on the A ring are disclosed in U.S. Pat. Nos. 3,622,627and 3,824,285. These oxime derivatives have hydrogen and halogen assubstituents at the C7 position and include7-halo-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline, and7-halo-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline.

Alkylamino (NH-alkyl), and alkylhydrazone (N-NH-alkyl) groups have beensubstituted on the A ring at the C4 position of4-dedimethylaminotetracycline. These compounds are known for theirantimicrobial properties. See U.S. Pat. Nos. 3,345,370, 3,609,188,3,622,627, 3,824,285, 3,622,627, 3,502,660, 3,509,184, 3,502,696,3,515,731, 3,265,732, 5,122,519, 3,849,493, 3,772,363, and 3,829,453.

In addition to their antimicrobial properties, tetracyclines have beendescribed as having a number of other uses. For example, tetracyclinesare also known to inhibit the activity of collagen destructive enzymes,such as matrix metalloproteinases (MMP), including collagenase (MMP-1),gelatinase (MMP-2) and stromelysin (MMP-3). Golub et al., J. Periodont.Res. 20:12-23 (1985); Golub et al. Crit. Revs. Oral Biol. Med. 2:297-322 (1991); U.S. Pat. Nos. 4,666,897; 4,704,383; 4,935,411;4,935,412. Also, tetracyclines have been known to inhibit wasting andprotein degradation in mammalian skeletal muscle, U.S. Pat. No.5,045,538, and to enhance IL-10 production in mammalian cells.

Furthermore, tetracyclines were reported to enhance bone proteinsynthesis in U.S. Pat. No. Re. 34,656, and to reduce bone resorption inorgan culture in U.S. Pat. No. 4,704,383.

Similarly, U.S. Pat. No. 5,532,227 to Golub et al, discloses thattetracyclines can ameliorate the excessive glycosylation of proteins. Inparticular, tetracyclines inhibit the excessive collagen cross linkingwhich results from excessive glycosylation of collagen in diabetes.

Tetracyclines are known to inhibit excessive phospholipase A₂ activityinvolved in inflammatory conditions such as psoriasis as disclosed inU.S. Pat. No. 5,532,227. In addition, tetracyclines are also known toinhibit cycloxygenase-2 (COX-2), tumor necrosis factor (TNF), nitricoxide and IL-1 (interleukin-1).

These properties cause the tetracyclines to be useful in treating anumber of diseases. For example, there have been a number of suggestionsthat tetracyclines, including non-antimicrobial tetracyclines, areeffective in treating arthritis. See, for example, Greenwald, et al.“Tetracyclines Suppress Metalloproteinase Activity in Adjuvant Arthritisand, in Combination with Flurbiprofen, Ameliorate Bone Damage,” Journalof Rheumatology 19:927-938(1992); Greenwald et al., “Treatment ofDestructive Arthritic Disorders with MMP Inhibitors: Potential Role ofTetracyclines in Inhibition of Matrix Metalloproteinases: TherapeuticPotential,” Annals of the New York Academy of Sciences 732: 181-198(1994); Kloppenburg, et al. “Minocycline in Active RheumatoidArthritis,” Arthritis Rheum 37:629-636(1994); Ryan et al., “Potential ofTetracycline to Modify Cartilage Breakdown in Osteoarthritis,” CurrentOpinion in Rheumatology 8: 238-247(1996); O'Dell et al, “Treatment ofEarly Rheumatoid Arthritis with Minocycline or Placebo,” Arthritis.Rheum. 40:842-848(1997).

Tetracyclines have also been suggested for use in treating skindiseases. For example, White et al., Lancet, Apr. 29, p. 966 (1989)report that the tetracycline minocycline is effective in treatingdystrophic epidermolysis bullosa, which is a life-threatening skincondition believed to be related to excess collagenase.

Furthermore, studies have also suggested that tetracyclines andinhibitors of metalloproteinases inhibit tumor progression, DeClerck etal., Annals N.Y. Acad. Sci., 732: 222-232 (1994), bone resorption,Rifkin et al., Annals N.Y. Acad. Sci., 732: 165-180 (1994),angiogenesis, Maragoudakis et al., Br. J. Pharmacol., 111: 894-902(1994), and may have anti-inflammatory properties, Ramamurthy et al.,Annals N.Y. Acad. Sci.,732, 427-430 (1994).

Based on the foregoing, tetracyclines have been found to be effective intreating numerous diseases and conditions. Therefore, there is a needfor new and even more useful 4-dedimethylaminotetracycline derivatives.

SUMMARY OF THE INVENTION

It has now been discovered that these and other objectives can beachieved by tetracycline compounds of the formulae:

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable andunacceptable salts thereof; with the following provisos: when either R7and R9 are hydrogen then R8 must be halogen; and when R6-a, R6, R5 andR9 are all hydrogen and R7 is hydrogen, amino, nitro, halogen,dimethylamino or diethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 and R9 are both hydrogen, R5 is hydroxyl and R7 is hydrogen,amino, nitro, halogen or diethylamino, then R8 is halogen; and when R6-ais methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 mustbe halogen; and when R6-a, R6 and R5 are all hydrogen, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6, R5and R9 are all hydrogen and R7 is cyano, then R8 must be halogen.

In another embodiment, the invention provides a tetracycline compound ofthe formulae:

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable and unacceptable salts thereof; with thefollowing provisos: when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a,R6, R5, and R9 are all hydrogen, then R8 must be halogen; and when R4 isNOH, R6-a is methyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 andR9 are both hydrogen, then R8 must be halogen; and when R4 isN—NH-alkyl, R6-a is methyl, R6 is hydroxyl and R7, R5, R9 are allhydrogen, then R8 must be halogen; and when R4 is NH-alkyl, R6-a, R6, R5and R9 are all hydrogen, R7 is hydrogen, amino, mono(lower alkyl)amino,halogen, di(lower alkyl)amino or hydroxyl, then R8 must be halogen; andwhen R4 is NH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 ishydroxyl, and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, thenR8 must be halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 ishydroxy or hydrogen and R7, R5, and R9 are all be hydrogen, then R8 mustbe halogen.

In yet another embodiment, the invention provides a 4-dedimethylaminotetracycline compound having general formulae (I) through (IV):

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azido hydrogenhydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium dimethylamino chloro amino hydrogen chloro aminoamino chloro amino acylamino chloro acylamino amino chloro hydrogenacylamino chloro hydrogen monoalkylamino chloro amino nitro chloro aminodimethylamino chloro acylamino dimethylamino chloro dimethylaminodimethylamino hydrogen hydrogen hydrogen hydrogen dimethylaminoand

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azido hydrogenhydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino hydrogenhydrogen diazonium hydrogen hydrogen dimethylamino diazonium hydrogenhydrogen ethoxythiocarbonylthio hydrogen hydrogen dimethylamino chloroamino amino chloro amino acylamino chloro acylamino hydrogen chloroamino amino chloro hydrogen acylamino chloro hydrogen monoalkyl aminochloro amino nitro chloro aminoand

wherein R8 is hydrogen or halogen and R9 is selected from the groupconsisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 amino hydrogen hydrogen nitro hydrogen hydrogen azido hydrogenhydrogen dimethylamino hydrogen azido hydrogen hydrogen amino hydrogenhydrogen azido hydrogen hydrogen nitro bromo hydrogen hydrogendimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino diethylamino hydrogenhydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylamino hydrogenmethylamino dimethylamino hydrogen acylamino dimethylamino chloro aminoamino chloro amino acylamino chloro acylamino hydrogen chloro aminoamino chloro hydrogen acylamino chloro hydrogen monoalkylamino chloroamino nitro chloro aminoand pharmaceutically acceptable and unacceptable salts thereof.

In yet another embodiment, the invention provides a tetracyclinecompound of the formulae:

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(c) andR^(d) are, independently (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and R^(e) is as above, NH,N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄) straightchain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof.

In a further embodiment, the following provisos apply: when either R7and R9 are hydrogen then R8 must be halogen; and when R6-a, R6, R5 andR9 are all hydrogen and R7 is hydrogen, amino, nitro, halogen,dimethylamino or diethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 and R9 are both hydrogen, R5 is hydroxyl, and R7 is hydrogen,amino, nitro, halogen or diethylamino, then R8 is halogen; and when R6-ais methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 mustbe halogen; and when R6-a, R6 and R5 are all hydrogen, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6, R5and R9 are all hydrogen and R7 is cyano, then R8. must be halogen.

In another embodiment, the tetracycline compounds described above andbelow preferably have a PIF value from about 1 to about 2, morepreferably at about 1. Some examples of tetracycline compounds having aPIF value of about 1 include:STRUCTURE Kwherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamideandSTRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoandSTRUCTURE Pwherein R8, and R9 are, respectively, hydrogen and nitro.

Some examples of tetracycline compounds having a PIF value from about 1to about 2 have the general formula:STRUCTURE K:wherein: R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino.

In another embodiment, the present invention provides a tetracyclinecompound of the formulae:STRUCTURE C STRUCTURE D STRUCTURE E STRUCTURE Fwherein R7 is selected from the group consisting of aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;and pharmaceutically acceptable and unacceptable salts thereof;

orSTRUCTURE C STRUCTURE D STRUCTURE E STRUCTURE Fwherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

orSTRUCTURE C STRUCTURE D STRUCTURE E STRUCTURE Fwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl alkynyl, or mixures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof.

In another embodiment, the invention provides a tetracycline compound ofthe formulae:STRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen;R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;

orSTRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of an aryl, alkenyl and alkynyl; and pharmaceuticallyacceptable and unacceptable salts thereof;

orSTRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; or mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R4 is selected from the groupconsisting of NOH, N—NH-A, and NH-A, where A is a lower alkyl group; andR8 is selected from the group consisting of hydrogen and halogen; andpharmaceutically acceptable and unacceptable salts thereof;

In another embodiment, the invention provides a tetracycline compound ofthe formulae:STRUCTURE Kwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;

orSTRUCTURE Kwherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

orSTRUCTURE Kwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; and R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof;andSTRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein: R7 is selected from the group consisting of an aryl, alkenyland alkynyl; R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;orSTRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

orSTRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein R7 is and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;andSTRUCTURE Pwherein R9 is selected from the group consisting of an aryl, alkenyl andalkynyl; and R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;andSTRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;

orSTRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

orSTRUCTURE Q STRUCTURE Rwherein R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof.

In another embodiment, the invention provides a tetracycline compound ofthe formulae:STRUCTURES S-Zwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;R^(a) and R^(b) are selected from the group consisting of hydrogen,methyl, ethyl, n-propyl and 1-methylethyl with the proviso that R^(a)and R^(b) cannot both be hydrogen; R^(c) and R^(d) are, independently,(CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) is selected from thegroup consisting of hydrogen, alkyl, hydroxy, lower(C₁-C₃) alkoxy,amino, or nitro; and, W is selected from the group consisting of(CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above, NH, N(C₁-C₃)straight chained or branched alkyl, O, S and N(C₁-C₄) straight chain orbranched alkoxy; and pharmaceutically acceptable and unacceptable saltsthereof;

orSTRUCTURES S-Zwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; R^(a) and R^(b) are selected from the group consisting ofhydrogen, methyl, ethyl, n-propyl and 1-methylethyl with the provisothat R^(a) and R^(b) cannot both be hydrogen; R^(c) and R^(d) are,independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) isselected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above,NH, N(C₁-C₃) straight chained or branched alkyl, 0, S and N(C₁-C₄)straight chain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof;

orSTRUCTURES S-Zwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; R^(a) and R^(b) are selected fromthe group consisting of hydrogen, methyl, ethyl, n-propyl and1-methylethyl with the proviso that R^(a) and R^(b) cannot both behydrogen; R^(c) and R^(d) are, independently, (CH₂)_(n)CHR^(e) wherein nis 0 or 1 and R^(e) is selected from the group consisting of hydrogen,alkyl, hydroxy, lower(C₁-C₃) alkoxy, amino, or nitro; and W is selectedfrom the group consisting of (CHR^(e))_(m) wherein m is 0-3 and saidR^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl, O, Sand N(C₁-C₄) straight chain or branched alkoxy; and pharmaceuticallyacceptable and unacceptable salts thereof.

The present invention includes a method for treating a mammal sufferingfrom a condition that benefits from a non-antimicrobial dose of atetracycline compound. Some examples of such conditions include thosecharacterized by excessive collagen destruction, excessive MMP enzymeactivity, excessive TNF activity, excessive nitric oxide activity,excessive IL-1 activity, excessive elastase activity, excessive loss ofbone density, excessive protein degradation, excessive muscle wasting,excessive glycosylation of collagen, excessive COX-2 activity,insufficient bone protein. synthesis, insufficient interleukin-10production, or excessive phospholipase A₂ activity. The method fortreating comprises administering to the mammal an effective amount of atetracycline compound of the invention.

Conditions that benefit from a non-antimicrobial dose of a tetracyclinecompound include, but are not limited to, abdominal aortic aneurysm,ulceration of the cornea, periodontal disease, diabetes, diabetesmellitus, scleroderna, progeria, lung disease, cancer, graft versus hostdisease, disease of depressed bone marrow function, thrombocytopenia,prosthetic joint loosening, spondyloarthropathies, osteoporosis, Paget'sdisease, autoimmune disease, systemic lupus erythematosus, acute orchronic inflammatory condition, renal disease, connective tissue diseaseor neurological and neurodegenerative conditions.

Acute or chronic inflammatory conditions that can benefit from anon-antimicrobial dose of a tetracycline compound can be, but are notlimited to, inflammatory bowel disease, arthritis, osteoarthritis,rheumatoid arthritis, pancreatitis, nephritis, glomerulonephritis,sepsis, septic shock, lipopolysaccharide endotoxin shock, multisystemorgan failure or psoriasis.

The lung diseases that can benefit from a non-antimicrobial dose of atetracycline compound can be, but are not limited to, ARDS, cysticfibrosis, emphysema or acute lung injury resulting from inhalation oftoxicants.

The renal diseases that can benefit from a non-antimicrobial dose of atetracycline compound include, but are not limited to chronic renalfailure, acute renal failure, nephritis or glomerulonephritis.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples which are set forth herein.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the photoirritancy factor (PIF), also known as thephotoinhibition factor, for some tetracycline compounds. For structureK, the compounds indicated are as follows:

COL R7 R8 R9 308 hydrogen hydrogen amino 311 hydrogen hydrogenpalmitamide 306 hydrogen hydrogen dimethylamino

For structures L, M, N or O the compounds indicated are as follows:

COL R7 R8 R9 801 hydrogen hydrogen acetamido 802 hydrogen hydrogendimethylaminoacetamido 804 hydrogen hydrogen nitro 805 hydrogen hydrogenaminoFor structure P, R7 is hydrogen, R8 is hydrogen and R9 is nitro.

DETAILED DESCRIPTION OF THE INVENTION

Particularly preferred compounds of the present invention have D ringsubstituents at the 7 and/or 9 positions on the4-dedimethylaminotetracycline molecule. These compounds include7-azido-6-demethyl-6-deoxy-4-dedimethylamino tetracycline,7-dimethylamino-9-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline, 9-amino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-amino-9-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(N,N,-dimethyl)glycylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7,9-diamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-ethoxythiocarbonylthio-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-dimethylamino-9-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-dimethylamino-9-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-amino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-nitro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-amino-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-amino-9-nitro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7, 9-diamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-dimethylamino-9-amino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-ethoxythiocarbonylthio-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-dimethylamino-9-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-azido-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-amino-8-chloro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,9-nitro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-acetamido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(N,N-dimethyl)glycylamino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,and9-ethoxythiocarbonylthio-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline.

In addition, the D ring may be halogenated at the C8 position to provide8-halodedimethylaminotetracycline derivatives. As used in thisspecification, halogens can be chlorine, fluorine, bromine, and iodine.Some examples of 8-halo-dedimethylaminotetracycline derivatives are9-amino-8-chloro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-amino-8-chloro-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracyclineand 9-amino-8-chloro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline.

In one embodiment of the invention, the 4-dedimethylaminotetracyclinederivatives are substituted with an oxime, NH-alkyl, or N—NH-alkyl groupat the C4 position. These compounds have the general formula:STRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, and di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable and unacceptable salts thereof; with thefollowing provisos: when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a,R6, R5, and R9 are all hydrogen, then R8 must be halogen; and when R4 isNOH, R6-a is methyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 andR9 are both hydrogen, then R8 must be halogen; and when R4 isN—NH-alkyl, R6-a is methyl, R6 is hydroxyl and R7, R5, R9 are allhydrogen, then R8 must be halogen; and when R4 is NH-alkyl, R6-a, R6, R5and R9 are all hydrogen, R7 is hydrogen, amino, mono(lower alkyl)amino,halogen, di(lower alkyl)amino or hydroxyl, then R8 must be halogen; andwhen R4 is NH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 ishydroxyl, and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, thenR8 must be halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 ishydroxy or hydrogen and R7, R5, and R9 are all be hydrogen, then R8 mustbe halogen.

It will be understood that if the stereochemistry of a substituent onrings A-D of the novel 4-dedimethylaminotetracycline derivative is notspecified, then both epimers are intended to be encompassed.

As used herein, NH-Alkyl, N—NH-Alkyl, alkoxy and alkyl groups containstraight or branched, saturated or unsaturated alkyl carbon chains,having from one to twenty-six carbon atoms. For example, alkyl groupsinclude fatty alkyls which contain ten to twenty-six carbon atoms. Someexamples of saturated fatty alkyl groups include, lauryl, myristyl,palmityl, stearyl, etc. Some examples of unsaturated fatty alkyl groupsinclude palmitoleyl, oleyl, linoleyl, linolenyl, etc.

Alkyl groups also include lower alkyls which include straight orbranched, saturated or unsaturated carbon chains, having from one to sixcarbon atoms. Some examples of lower alkyl groups are methyl, ethyl,propyl, butyl, isobutyl, n-butyl, secondary butyl, tertiary butyl,n-pentyl and benzyl. The alkyl moiety of acyl groups is defined asabove. Some examples of acyl groups include acetyl, propionyl, butyryl,and acyl groups comprising fatty acids such as those described above.

Preferred oxime compounds include7-azido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-dimethylamino-9-azido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-amino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-azido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-nitro-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-acetamido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-acetamido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-amino-9-nitro-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-(N,N,-dimethyl)glycylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7,9-diamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-ethoxythiocarbonylthio-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-dimethylamino-9-acetamido-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-azido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-dimethylamino-9-azido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximino-tetracycline,9-amino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-azido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-nitro-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-amino-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-acetamido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-acetamido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-amino-9-nitro-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,also known as9-dimethylaminoacetamido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7,9-diamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-dimethylamino-9-amino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-ethoxythiocarbonylthio-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,7-dimethylamino-9-acetamido-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-azido-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-amino-8-chloro-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-nitro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-acetamido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximino-tetracycline,9-(N,N-dimethyl)glycylamino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximino-tetracycline,and9-ethoxythiocarbonylthio-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline.

In addition, the D ring may be halogenated at the C8 position to provide8-halo-4-dedimethylamino-4-oximinotetracycline compounds. Some examplesinclude9-amino-8-chloro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline,9-amino-8-chloro-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylamino-4-oximinotetracyclineand9-amino-8-chloro-6-demethyl-6-deoxy-4-dedimethylamino-4-oximinotetracycline.

Preferred hydrazone compounds include7-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,7-dimethylamino-9-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-amino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,7-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,7-amino-9-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-(N,N,-dimethyl)glycylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone, 7,9-diamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-ethoxythiocarbonylthio-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,7-dimethylamino-9-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl or ethyl hydrazone,9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,7-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,7-dimethylamino-9-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-amino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-azido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone, 9-nitro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl or ethyl hydrazone,9-amino-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,7-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,9-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,7-amino-9-nitro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone, 9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone, 7,9-diamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl orethyl hydrazone,7-dimethylamino-9-amino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-ethoxythiocarbonylthio-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,7-dimethylamino-9-acetamido-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-azido-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-amino-8-chloro-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-(N,N-dimethyl)glycylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methyl or ethyl hydrazone,9-nitro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-acetamido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone,9-(N,N-dimethyl)glycylamino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone, and9-ethoxythiocarbonylthio-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone.

The D ring may be halogenated at the C8 position to provide8-halo-4-dedimethylaminotetracycline-4-hydrazone compounds. Someexamples include9-amino-8-chloro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methyl or ethyl hydrazone,9-amino-8-chloro-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone and9-amino-8-chloro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline-4-methylor ethyl hydrazone.

Novel 4-dedimethylaminotetracycline derivatives of the present inventionalso include compounds with an NH-Alkyl (alkylamino) substituent at theC4 position on the A ring. These compounds have substitutions at the C5,C6, C6a, C7, C8 and/or C9 positions as described above. An example is9-azido-8-chloro-7-dimethylamino-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline.

In addition, a hexanoylamino group can be added to the C9 position onthe D ring of any compound of the invention. An example includes, but isnot limited to,4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline.

In another embodiment of the invention, the4-dedimethylaminotetracycline derivatives are Mannich derivatives of thecompounds described above. Such derivatives include, for example,compounds having the general formula:STRUCTURE S STRUCTURE T STRUCTURE U STRUCTURE VSTRUCTURE W STRUCTURE X STRUCTURE Y STRUCTURE Zwherein R5, R6, R7, R8 and R9 are as described above; R^(a) and R^(b)are selected from the group consisting of hydrogen, methyl, ethyl,n-propyl or 1-methylethyl with the proviso that R^(a) and R^(b) cannotboth be hydrogen; R^(c) and R^(d) are, independently, (CH₂)_(n)CHR^(e)wherein n is 0 or 1 and R^(e) is selected from the group consisting ofhydrogen, alkyl, hydroxy, lower(C₁-C₃) alkoxy, amino, or nitro; and, Wis selected from the group consisting of (CHR^(e))_(m) wherein m is 0-3and R^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl,O, S and N(C₁-C₄) straight chain or branched alkoxy; andpharmaceutically acceptable and unacceptable salts thereof. For example,when m is 0, R^(c) and R^(d) are bonded to each other in a 3-5 memberedring, such as, for example, a pyrrolidino or substituted pyrrolidinoring, a morpholino or substituted morpholino ring, or a piperazino orsubstituted piperazino ring.

These Mannich derivatives include, for example, compounds with apiperazin-1-yl, 4-methylpiperazin-1-yl, morpholin-1-yl, orpyrrolidin-1-yl substituent at the C2 position. These compounds havesubstituents at the C4, C5, C6, C6a, C7, C8 and/or C9 positions asdescribed above. Examples of such compounds include, but are not limitedto,N-morpholin-1-ylmethyl-4-dedimethylamino-6-demethyl-6-deoxytetracyline,N-pyrrolidin-1-ylmethyl-4-dedimethylamino-6-demethyl-6-deoxytetracyline,N-morpholin-1-ylmethyl-4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotertracycline,N-pyrrolidin-1-ylmethyl-4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline.

In another embodiment of the invention, the4-dedimethylaminotetracycline derivatives are substituted at the C7 orC9 position, or at both the C7 and C9 positions, with an aryl, alkenyl,or alkynyl group, or mixtures thereof. Such compounds include, forexample, compounds having any of the general formulas C-Z wherein R4,R5, R6, R6a, R8, Ra, Rb, Rc, Rd and W are as described above; with theprovisos that when R7 is not an aryl, alkenyl or alkynyl group, R7 is ahydrogen, amino, nitro, mono(lower alkyl)amino, halogen, di(loweralkyl)amino, ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano,or hydroxyl; and when R9 is not an aryl, alkenyl or alkynyl group, R9 ishydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino or RCH(NH₂)CO.

In this specification, an aryl group is any monocyclic or polycyclicaromatic group derived from an aromatic compound. The most typicalexample of a monocyclic aromatic compound is benzene and substitutedbenzene derivatives. Examples of polycyclic aromatic compounds include,but are not limited to, naphthalene, anthracene, 1,2-benzylpyrene, andcoronene.

The alkenyl, and alkynyl groups at either or both of the C7 or C9positions include any of the alkyl groups described herein, furtherhaving, respectively, one or more double or triple bonds, preferably oneto three double or triple bonds, at any position. Some examples ofalkenyl groups include, but are not limited to, ethylenyl, propenyl,1-butylenyl, 2-butylenyl and 2-methylpropylenyl and 1,3 hexadienyl. Thealkynyl groups include, but are not limited to, ethynyl, propynyl,1-butynyl, 2-butynyl and 2-methylpropynyl and 1,3 hexadiynyl.

The aryl, alkenyl and alkynyl groups may be further substituted at anyposition with one or more additional substituents. Some examples of suchfurther substitutions include, but are not limited to, nitro, amino,halo (F, CI, Br or I), amido, azido, cyano, hydroxyl, alkoxy, preferablylower alkoxy, acyl, preferably lower acyl, amidoazido, mono(loweralkyl)amino, di(lower alkyl)amino, ethoxythiocarboxylthio, diazonium,acylamino, N, N-dimethylglycylamino, and alkyl groups, preferably loweralkyl groups. Examples of substituted benzene derivatives include, butare not limited to, methylbenzene (toluene), nitrobenzene,hydroxybenzene (phenol), aminobenzene (aniline), vinylbenzene (styrene),benzaldehyde, benzoic acid, 1,2-dimethylbenzene (ortho-xylene), 1,3dimethylbenzene (meta-xylene), and 1,4-dimethylbenzene (para-xylene).

Some examples of compounds having aryl substituents at the C7 or C9positions include7-phenyl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-phenyl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-(4-fluorophenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(4-fluorphenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-(4-chlorophenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(4-chlorophenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-(4-nitrophenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(4-nitrophenyl)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-(4-dimethylamino)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,9-(4-dimethylamino)-6-demethyl-6-deoxy-4-dedimethylaminotetracycline,7-phenyl-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,9-phenyl-5-hydroxy-6-deoxy-4-dedimethylaminotetracycline,7-(4-fluorophenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,9-(4-fluorphenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,7-(4-chlorophenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,9-(4-chlorophenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,7-(4-nitrophenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,9-(4-nitrophenyl)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,7-(4-dimethylamino)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,9-(4-dimethylamino)-5-hydroxy-6-deoxy-4-dedimethyaminotetracycline,7-phenyl-7-dimethylamino-4-dedimethylaminotetracycline,9-phenyl-7-dimethylamino-4-dedimethylaminotetracycline,7-(4-fluorophenyl)-7-dimethylamino, 4-dedimethylaminotetracycline,9-(4-fluorphenyl)-7-dimethylamino-4-dedimethylaminotetracycline,7-(4-chlorophenyl)-7-dimethylamino-4-dedimethylaminotetracycline,9-(4-chlorophenyl)-7-dimethylamino-4-dedimethylaminotetracycline,7-(4-nitrophenyl)-7-dimethylamino-4-dedimethylaminotetracycline,9-(4-nitrophenyl)-7-dimethylamino-4-dedimethylaminotetracycline,7-(4-dimethylamino)-7-dimethylamino-4-dedimethylaminotetracycline,9-(4-dimethylamino)-7-dimethylamino-4-dedimethylaminotetracycline,7-phenyl-4-dedimethylaminotetracycline,9-phenyl-4-dedimethylaminotetracycline,7-(4-fluorophenyl)-4-dedimethylaminotetracycline,9-(4-fluorphenyl)-4-dedimethylaminotetracycline,7-(4-chlorophenyl)-4-dedimethylaminotetracycline,9-(4-chlorophenyl)-4-dedimethylaminotetracycline,7-(4-nitrophenyl)-4-dedimethylaminotetracycline,9-(4-nitrophenyl)-4-dedimethylaminotetracycline,7-(4-dimethylamino)-4-dedimethylaminotetracycline,9-(4-dimethylamino)-4-dedimethylaminotetracycline.

Some examples of compounds having alkenyl or alkynyl substituents at theC7 or C9 positions include the same C7 and C9 aryl substitutedtetracycline derivatives described above, but instead having alkenyl oralkynyl substituents that are two to four carbons in length attached tothe C7 and C9 positions. The alkenyl groups include, but are not limitedto, ethenyl, propenyl, 1-butenyl, 2-butenyl and 2-methylpropenyl. Thealkynyl groups include, but are not limited to, ethynyl, propynyl,1-butynyl, 2-butynyl and 2-methylpropynyl.

The novel 4-dedimethylaminotetracycline compounds of the presentinvention including pharmaceutically acceptable and unacceptable saltsthereof may be prepared by D ring substitution at the C7, C8 and/or C9positions using starting reactants that can readily be prepared orpurchased by methods known in the art. See, for example, Mitscher, L.A., The Chemistry of the Tetracycline Antibiotics, Marcel Dekker, NewYork (1978), Ch. 6, Hlavka, J. and J. H. Boothe, The Tetracyclines,Springer-Verlag, Berlin-Heidelberg, page 18 (1985) and U.S. Pat. Nos.4,704,383, 3,226,436, 3,047,626 3,518,306 and 5,532,227.

For example, nitration of the C9 position on the D ring may beaccomplished, and novel 9-nitro compounds may be prepared, by usingknown starting reactants such as7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline or6-deoxy-4-dedimethylaminotetracycline and treating these compounds witha strong acid and metal nitrate salts. Examples of strong acids that aresuitable for use in the present invention are: sulfuric acid,trifluoroacetic acid, methanesulfonic acid or perchloric acid. Suitablemetal nitrate salts are, for example, calcium, potassium or sodiumnitrate. The C9 position on the D ring undergoes nitration to form thecorresponding9-nitro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-nitro-6-deoxy-4-dedimethylaminotetracycline compounds.

Amination of the C9 position on the D ring may be accomplished bytreating a 9-nitro-4-dedimethylaminotetracycline, such as9-nitro-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-nitro-6-deoxy-4-dedimethylaminotetracycline with hydrogen in thepresence of a suitable supported catalyst such as Raney nickel, platinumoxide or palladium-on-carbon. This is then filtered and washed with anorganic solvent such as ether. The C9 substituent is reduced to form thecorresponding9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-amino-6-deoxy-4-dedimethylaminotetracycline compound.

The amino group on the D ring at the C9 position may be converted to anacylamido group or a sulfonamido group. For example,9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-amino-6-deoxy-4-dedimethylaminotetracycline compounds are treatedwith acyl chloride, acyl anhydride, mixed acyl anhydride, sulfonylchloride or sulfonyl anhydride in the presence of a suitable acidscavenger dispersed in a solvent. The acid scavenger is suitablyselected from sodium bicarbonate, sodium acetate, pyridine,triethylamine, N,O-bis(trimethylsilyl)acetamide,N,O-bis(trimethylsilyl)trifluoroacetamide or a basic ion-exchange resin.Solvents suitable for the acylation reaction include water,water-tetrahydrofluran, N-methylpyrolidone,1,3-dimethyl-2-imidazolidione, hexamethylphosphoramide,1,3,dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone or1,2-dimethoxyethane. The C9 amino group may be converted to theacetamido group to form, for example, the corresponding9-acetamido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline or 9-acetamido-6-deoxy-4-dedimethylamino tetracycline.

A diazonium group can also be substituted at the C9 position on theD-ring. Typically, a 9-amino-4-dedimethylaminotetracycline derivative,such as9-amino-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-amino-6-deoxy-4-dedimethylaminotetracycline in 0.1N HCL in methanolis treated with n-butyl nitrite to form the corresponding 9-diazoniumderivatives such as9-diazonium-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-diazonium-6-deoxy-4-dedimethylaminotetracycline.

The 9-diazonium-4-dedimethylaminotetracycline derivatives, such as9-diazonium-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-diazonium-6-deoxy-4-dedimethylaminotetracycline can be treated withmethanolic hydrochloric acid plus a triazo compound such as sodium azideto form, 9-azido derivatives, such as9-azido-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineor 9-azido-6-deoxy-4-dedimethylaminotetracycline.

Alternately, an ethoxythiocarbonylthio group can be substituted at theC9 position on the D ring. For example, a9-diazonium-4-dedimethylaminotetracycline derivative, such as9-diazonium-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline or 9-diazonium-6-deoxy-4-dedimethylaminotetracycline istreated with an acid metal salt such as potassium ethyl xanthate to formthe corresponding 9-ethoxythiocarbonylthio derivative, such as9-ethoxythiocarbonylthio-7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminoor 9-ethoxythiocarbonylthio-6-deoxy-4-dedimethylaminotetracycline.

The above reactions describe substitution at the C9 position on4-dedimethylaminotetracycline molecule. Some substitution may alsooccur, depending on the starting reactants and conditions used, at theC7 position and lead to also 7-substituted-4-dedimethylaminotetracyclinederivatives, such as7-diazonium-6-demethyl-6-deoxy-4-dedimethylaminotetracycline or7-azido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline. The7-substituted derivatives can be separated from the 9-substitutedderivatives, and purified as discussed below.

The novel 7 or 9 azido-4-dedimethylamino derivatives of the presentinvention can be halogenated at the C8 position by treating 7 or9-azido-4-dedimethylaminotetracycline with a strong acid such ashydrogen halide, sulfuric acid saturated with hydrogen halide ormethanesulfonic acid saturated with hydrogen halide. The product that isisolated, when the hydrogen halide is hydrogen chloride, is the 8-chloro(7 or 9) amino-4-dedimethylaminotetracycline derivative. A particularlypreferred halogenated compound is9-acetamido-8-chloro-7-dimethylamino-6-deoxy-6-demethyl-4-dedimethylaminotetracycline.

In one embodiment, 4-dedimethylaminotetracycline compounds of thepresent invention have an oxime (NOH), alkylamino (NH-alkyl), oralkylhydrazone (N—NH-alkyl) group at the C4 position on the A ring.These compounds can be made using known methods. For example,4-hydroxytetracycloxide may be treated with hydroxyamine orethylhydrazine under alkaline conditions in a solvent such as methanolor ethanol. Substitution at the C4 position occurs and4-dedimethylamino-4-oximinotetracycline and4-dedimethylaminotetracycline-4-alkylhydrazone compounds can be isolatedas alkali metal salts. See for example, U.S. Pat. Nos. 3,622,627,3,159,675 and 3,345,370. Substitution at C7, C8, and/or C9 positions onthe D ring using methods previously described (i.e. halogenation,amination, or nitration) give rise to the novel 4-oxime, 4-hydrazone and4-aminoalkyl compounds of the present invention.

The Mannich derivatives can be made by methods known in the art. Forexample, the tetracycline derivatives described above may be treatedwith formaldehyde and the appropriate amine.

Aryl, alkenyl and alkynyl groups can be added onto the C7 and C9positions of the tetracycline derivatives described above by methodsknown in the art. Such methods include modified Suzuki and Stillecoupling reactions. See, for example, Koza D. J., Organic Letters, 2000.For example, the tetracycline derivatives described above having aniodine attached to the C7or C9 positions can be treated withtri-n-butylstannyl reagent in the presence of a palladium catalyst andcopper iodide. This reaction can be extended to a variety of aryl,alkenyl and alkynyl derivatives.

Examples of specific embodiments are described above as derivatives ofthe specific antibiotic compound tetracycline. The compounds of theinvention are not, however, limited to derivatives of any specifictetracycline compound. Rather, the compounds of the invention includethe 4-dedimethyl derivatives of any member of the tetracycline family.Thus, the invention also includes, but is not limited to, the same4-dedimethylamino derivatives and 4-substituted 4-dedimethylaminoderivatives of sancycline, minocycline, and doxycycline as thetetracycline derivatives mentioned above.

The present invention embraces salts, including acid-addition and metalsalts, of the 4-dedimethylaminotetracycline compounds described herein.Such salts are formed by well known procedures with bothpharmaceutically acceptable and pharmaceutically unacceptable acids andmetals. By “pharmaceutically acceptable” is meant those salt-formingacids and metals which do not substantially increase the toxicity of thecompound.

Some examples of suitable salts include salts of mineral acids such ashydrochloric, hydriodic, hydrobromic, phosphoric, metaphosphoric, nitricand sulfuric acids, as well as salts of organic acids such as tartaric,acetic, citric, malic, benzoic, glycollic, gluconic, gulonic, succinic,arylsulfonic, e.g. p-toluenesulfonic acids, and the like. Thepharmaceutically unacceptable acid addition salts, while not useful fortherapy, are valuable for isolation and purification of the newsubstances. Further, they are useful for the preparation ofpharmaceutically acceptable salts. Of this group, the more common saltsinclude those formed with hydrofluoric and perchloric acids.Hydrofluoride salts are particularly useful for the preparation of thepharmaceutically acceptable salts, e.g. the hydrochlorides, bydissolution in hydrochloric acid and crystallization of thehydrochloride salt formed. The perchloric acid salts are useful forpurification and crystallization of the new products.

Whereas metal salts may, in general, be prepared and are useful forvarious purposes, the pharmaceutically acceptable metal salts areparticularly valuable because of their utility in therapy. Thepharmaceutically acceptable metals include more commonly sodium,potassium and alkaline earth metals of atomic number up to and including20, i.e., magnesium and calcium and additionally, aluminum, zinc, ironand manganese, among others. Of course, the metal salts include complexsalts, i.e. metal chelates, which are well recognized in thetetracycline art.

After preparation, the novel compounds of the present invention can beconveniently purified by standard methods known in the art. Somesuitable examples include crystallization from a suitable solvent orpartition-column chromatography.

The novel 4-dedimethylaminotetracycline compounds of the presentinvention can be used in vivo, in vitro, and ex vivo, for example, inliving mammals as well as in cultured tissue, organ or cellular systems.Mammals include, for example, humans, as well as pet animals such asdogs and cats, laboratory animals, such as rats and mice, and farmanimals, such as horses and cows. Tissues, as used herein, are anaggregation of similarly specialized cells which together performcertain special functions. Cultured cellular systems include anymammalian cells, such as epithelial, endothelial, red blood, and whiteblood cells. More particularly, human peripheral blood monocytes,synovial fibroblastoid cells, and the like.

The present invention is directed to a method for treating a mammalsuffering from a condition or diseases that benefits from anon-antimicrobial dose of a tetracycline compound. These conditions ordiseases are characterized by excessive collagen destruction, excessiveMMP enzyme activity, excessive TNF activity, excessive nitric oxideactivity, excessive IL-1 activity, excessive elastase activity,excessive loss of bone density, excessive protein degradation, excessivemuscle wasting, excessive glycosylation of collagen, excessive COX-2activity, insufficient bone protein synthesis, insufficient IL-10(interleukin-10) production or excessive phospholipase A₂ activity. Themethod comprises administering to the mammal an effective amount of atetracycline compound of the invention.

The term “excessive,” as used herein, refers to increased activity overusual activity which leads to some pathological problem in a mammal ormammalian cells.

In vivo practice of the invention permits application in the relief orpalliation of medical and veterinary diseases, conditions, andsyndromes. In particular, the present invention includes a method fortreating a mammal suffering from conditions or diseases includingabdominal aortic aneurysm, ulceration of the cornea, periodontaldisease, diabetes, diabetes mellitus, scleroderma, progeria, lungdisease, cancer, graft versus host diseases, disease of depressed bonemarrow function, thrombocytopenia, prosthetic joint loosening,spondyloarthropathies, osteoporosis, Paget's disease, autoimmunedisease, systemic lupus erythematosus, acute or chronic inflammatoryconditions, renal disease or connective tissue disease by administeringan effective amount of a tetracycline compound to the mammal.

Cancerous conditions treatable by tetracycline compounds of the presentinvention include, but are not limited to, carcinomas, blastomas,sarcomas such as Kaposi's Sarcoma, gliomas, and the twelve majorcancers: prostrate cancer, breast cancer, lung cancer, colorectalcancer, bladder cancer, non-Hodgkin's lymphoma, uterine cancer,melanoma, kidney cancer, leukemia, ovarian cancer and pancreatic cancer.

Acute or chronic inflammatory conditions treatable by tetracyclinecompounds of the present invention include, for example, inflammatorybowel disease, arthritis, osteoarthritis, rheumatoid arthritis,pancreatitis, nephritis, glomerulonephritis, sepsis, septic shock,lipopolysaccharide endotoxin shock, multisystem organ failure orpsoriasis.

Lung diseases treatable by means of the present invention include, forexample, ARDS (adult respiratory distress syndrome), cystic fibrosis,emphysema or acute lung injury resulting from inhalation of toxicants.Some examples of toxicants are acids, chemicals, industrial and militarypoisons, smoke and other toxic products of combustion.

The novel tetracycline compounds of the present invention can also beused to treat renal diseases. Some examples of renal diseases arechronic renal failure, acute renal failure, nephritis orglomerulonephritis.

The tetracycline compounds of the present invention can also be used totreat neurological and neurodegenerative conditions. Examples ofneurological and neurodegenerative conditions include, Alzheimer'sdisease, Guillain-Barré Syndrome (acute febrile polyneuritis), Krabbe'sdisease (leukodystrophy), adrenoleukodystrophy, Parkinson's disease,Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis(Lou Gehrig's disease) and encephalopathies, including spongiformencephalopathy, such as Creutzfeldt-Jakob Disease. Other examples ofspongiform encephalopathies in humans include kuru,Gerstmann-Straussler-Scheinker disease and fatal familial insomnia.Other examples of spongiform encephalopathies in mammals include bovinespongiform encephalopathy (mad cow disease), scrapie in sheep and goats,transmissible mink encephalopathy, chronic wasting disease of mule deerand elk, and feline spongiform encephalopathy.

An effective amount of a tetracycline compound as used herein is thatamount effective to achieve the specified result of treating the diseaseor condition. Preferably, the tetracycline compound or derivative isprovided in an amount which has little or no antimicrobial activity. Atetracycline compound or derivative is not effectively antimicrobial ifit does not significantly prevent the growth of microbes. Accordingly,the method can beneficially employ a tetracycline derivative which hasbeen modified chemically to reduce or eliminate its antimicrobialproperties. The use of such chemically-modified tetracyclines ispreferred in the present invention since they can be used at higherlevels than antimicrobial tetracyclines, while avoiding certaindisadvantages, such as the indiscriminate killing of beneficialmicrobes, and the emergence of resistant microbes, which oftenaccompanies the use of antimicrobial or antibacterial amounts of suchcompounds.

The maximal dosage for a mammal is the highest dosage which does notcause undesirable or intolerable side effects. Minimal dosage is thelowest dosage where efficacy is first observed. For example, thetetracycline compound can be administered in an amount of from about 0.1mg/kg/day to about 30 mg/kg/day, and preferably from about 1 mg/kg/dayto about 18 mg/kg/day. In any event, the practitioner is guided by skilland knowledge in the field, and the present invention includes withoutlimitation dosages which are effective to achieve the described effect.

The method involves administering or providing a tetracycline derivativein an amount which is effective for treating diseases or conditions inmammalian cells or a mammal. Administering the tetracycline derivativescan be accomplished in a variety of ways. In cultured cellular systems(in vitro), tetracycline derivatives can be administered by contactingthe cells directly with an effective amount of the tetracyclinederivative.

In living mammals (in vivo), tetracycline derivatives of the presentinvention can be administered systemically by the parenteral and enteralroutes which also includes controlled release delivery systems. Forexample, tetracycline derivatives of the present invention can easily beadministered intravenously (e.g., intravenous injection) which is apreferred route of delivery. Intravenous administration can beaccomplished by mixing the tetracycline derivatives in a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art.

Oral or enteral use is also contemplated, and formulations such astablets, capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like can be employed to provide the tetracyclinederivative.

Alternatively, delivery of the tetracycline derivative can includetopical application. Accordingly, the carrier is preferably suited fortopical use. Compositions deemed to be suited for such topical useinclude gels, salves, lotions, creams ointments and the like. Thetetracycline derivative may also be incorporated with a support base ormatrix or the like to provide a pre-packaged surgical or burn dressingor bandage which can be directly applied to skin. Topical application oftetracycline derivatives in amounts of up to about 25% (w/w) in avehicle are therefore appropriate depending upon indication. Morepreferably, application of tetracycline derivatives in amounts of fromabout 0.1% to about 10% is believed to be effective in treating diseasesor conditions. It is believed that these quantities do not inducesignificant toxicity in the subject being treated.

For example, in certain cases tetracycline compounds having only limitedbiodistribution may be preferred for localized activity. Topicalapplication of these non-absorbable CMTs would be desirable in orallesions, since the CMTs would not be absorbed to any significant degreeeven if swallowed.

Combined or coordinated topical and systemic administration oftetracycline derivatives is also contemplated under the invention. Forexample, a non-absorbable tetracycline compound can be administeredtopically, while a tetracycline compound capable of substantialabsorption and effective systemic distribution in a subject can beadministered systemically.

PHOTOTOXICITY

In one embodiment, the invention relates to a class of compounds thathave low phototoxicity. To identify potentially phototoxic tetracyclinederivatives, the 3T3 Neutral Red Phototoxicity assay was employed. Theassay is described in Toxicology In Vitro 12:305-327, 1998.

Briefly, 3T3 cells are seeded in to 96-well plates and incubated overnight. The growth medium is removed and replaced with phenol-red freeHanks' Balanced Salt Solution containing serial dilutions of thetetracycline derivatives (two plates per compound). After an initial onehour incubation at 37° C., one plate is exposed to 5 Joules/cm² ofUVA/white light from a solar simulator while the other is held in thedark. The plates are then rinsed, re-fed and incubated for 24 hours.Cell visibility is measured by neutral red uptake. Phototoxicity ismeasured by the relative toxicity between the doses with and withoutlight exposure following published guidelines. (Reference compoundsinclude commercially available tetracycline, doxycycline, andminocycline.) The relative phototoxicity is called photoirritancy factor(PIF). The phototoxic response of the compounds in the present assay isconsistent with their behavior in vivo.

The class of low phototoxicity tetracyline derivatives has less than 75%of the phototoxicity of minocycline, preferably less than 70%, morepreferably less than 60%, and most preferably 50% or less, wherein thephototoxicity of minocycline is about 2.04. Optimally, the class of lowphototoxicity tetracycline derivatives have PIF values of 1. At a PIFvalue of 1, a compound is considered to have no measurablephototoxicity. Members of this class include, but are not limited to,tetracycline compounds having general forumulae:STRUCTURE Kwherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamide hydrogenhydrogen dimethylaminoandSTRUCTURE L STRUCTURE MSTRUCTURE N STRUCTURE Owherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoSTRUCTURE Pwherein: R8, and R9 are, respectively, hydrogen and nitro.

The class of low phototoxicity tetracycline compound derivativesincludes those derivatives having PIF values greater than 1, i.e. 1 toabout 2, preferably 1 to about 1.5. One example is a tetracyclinederivative having the general formula:STRUCTURE K:wherein: R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Example 14-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-nitrotetracyclinesulfate

To a solution of one millimole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline in 25ml of concentrated sulfuric acid at 0° C. was added 1.05 mmole ofpotassium nitrate. The resulting solution was stirred at ice bathtemperature for 15 minutes and poured in one liter of cold ether withstirring. The precipitated solid was allowed to settle and the majorityof solvent decanted. The remaining material was filtered through asintered glass funnel and the collected solid was washed well with coldether. The product was dried in a vacuum desiccator overnight.

Example 29-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 300 mg of the 9-nitro compound from example 1, in 30 mlof ethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The productthat separates is filtered and dried in a vacuum desiccator.

Example 39-Acetamido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate from example 2, in 2.0 ml of 1.3-dimethyl-2-imidazolidinone, 500mg of sodium bicarbonate was added followed by 0.21 ml of acetylchloride. The mixture is stirred at room temperature for 30 minutes,filtered and the filtrate was added dropwise to 500 ml of ether. Theproduct that separated was filtered and dried in a vacuum desiccator.

Example 44-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate

To a solution of 0.5 g of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate, from example 2, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath, 0.5 ml of n-butyl nitrite was added. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 59-Azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 0.3 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, 10 ml of 0.1 N methanolic hydrogen chloride wasadded 0.33 m mole of sodium azide. The mixture was stirred at roomtemperature for 1.5 hours. The reaction mixture was then poured into 200ml of ether. The product that separated was filtered and dried in avacuum desiccator.

Example 69-Amino-8-chloro-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-tetracyclinesulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinehydrochloride, from example 4, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 74-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-ethoxythiocarbonylthio-tetracyclinesulfate

A solution of 1.0 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, in 15 ml of water was added to a solution of1.15 mmole of potassium ethyl xanthate in 15 ml of water. The mixturewas stirred at room temperature for one hour. The product separated andwas filtered and dried in a vacuum desiccator.

Example 8A General Procedure for Nitration

To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml ofconcentrated sulfuric acid at 0° C. was added 1 mmole of potassiumnitrate with stirring. The reaction solution was stirred for 15 minutesand then poured into 100 g of chopped ice. The aqueous solution wasextracted 5 times with 20 ml of butanol each time. The butanol extractswere washed three times with 10 ml of water each time, and concentratedin vacuo to a volume of 25 ml. The light yellow crystalline solid whichprecipitated was filtered, washed with 2 ml of butanol and dried invacuo at 60° C. for 2 hours. This solid was a mixture of the twomononitro isomers.

Example 8B 4-Dedimethylamino-6-deoxy-9-nitrotetracycline

To 980 mg of the nitration product from4-dedimethylamino-6-deoxytetracycline (a mixture of the 2 isomers) in 25ml of methanol was added enough triethylamine to dissolve the solid. Thefiltered solution (pH 9.0) was adjusted to pH 5.2 with concentratedsulfuric acid. A crystalline yellow solid (236 mg.) was obtained (29%yield). The material at this point was quite pure and contained onlysmall amounts of the 7-isomer. Final purification was accomplished byliquid partition chromatography using a diatomaceous earth packed columnand the solvent system: chloroform: butanol: 0.5 M phosphate buffer (pH2) (16:1:10).

Example 9 4-Dedimethylamino-6-deoxy-7-nitrotetracycline

The methanol filtrate from example 8 was immediately adjusted to pH 1.0with concentrated sulfuric acid. The light yellow crystalline solid,which was obtained as the sulfate salt. A purified free base wasobtained by adjusting an aqueous solution of the sulfate salt (25 mg/ml)to pH 5.2 with 2 N sodium carbonate.

Example 10 9-Amino-4-dedimethylamino-6-deoxytetracycline

To a solution of 300 mg of the 9-nitro compound, prepared in example 8,in 30 ml of ethanol was added 50 mg of PtO₂. The mixture washydrogenated at atmospheric pressure until the theoretical amount ofhydrogen was absorbed. The system is flushed with nitrogen, the PtO₂catalyst is filtered and the filtrate added dropwise to 300 ml of ether.The solid that separates is filtered and dried in a vacuum desiccator.

Example 11 9-Acetamido-4-dedimethylamino-6-deoxytetracycline sulfate

To well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-6-deoxytetracycline sulfate, from example 10,in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodiumbicarbonate followed by 0.21 ml of acetyl chloride. The mixture wasstirred at room temperature for 30 minutes, filtered and the filtratewas added dropwise to 500 ml of ether. The solid that separated wasfiltered and dried in a vacuum desiccator.

Example 12 4-Dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate

To a solution of 0.5 g of 9-amino-4-dedimethylamino-6-deoxytetracyclinesulfate, from example 10, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and the poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 13 9-Azido-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, of example12, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33 mmole ofsodium azide. The mixture was stirred at room temperature for 1.5 hours.The reaction mixture was then poured into 200 ml of ether. The solidthat separated was filtered and dried in a vacuum desiccator.

Example 14 9-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclinesulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracyclinehydrochloride, from example 13, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The solid that separated was filtered, washed and etherand dried in a vacuum desiccator.

Example 154-Dedimethylamino-6-deoxy-9-ethoxythiocarbonylthiotetracycline sulfate

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, from example12, in 15 ml of water was added to a solution of 1.15 mmole of potassiumethyl xanthate in 15 ml of water. The mixture was stirred at roomtemperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 16 9-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg. of the 9-amino compound from example 10, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml. of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is hydrogenated underatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 98 mg.

Example 17 7-Amino-4-dedimethylamino-6-deoxytetracycline

This compound can be made using Procedure A or B. Procedure A. To asolution of 300 mg of the 7-nitro compound, from example 1, in 30 ml ofethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The solidthat separates is filtered and dried in a vacuum desiccator.

Procedure B. 1 g of 6-deoxy-4-dedimethylamino-tetracycline was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g of dibenzyl azodicarboxylatewas added and the mixture stirred for 2 hours at 0° C. to yield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-4-dedimethylaminotetracycline.

Example 18 7-Amino-6-deoxy-5-hydroxy-4-dedimethylaminotetracycline

1 g of 6-deoxy-5-hydroxy-4-dedimethylaminotetracycline 3 was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g dibenzyl azodicarboxylate in0.5 ml THF was added and the mixture stirred for 2 hours at 0° C. toyield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxy-5-hydroxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-5-hydroxytetracycline.

Example 19 7-Acetamido-4-dedimethylamino-6-deoxy-5-hydroxytetracyclinesulfate

To well stirred cold solution of 500 mg of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 18, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mgof sodium bicarbonate followed by 0.21 ml of acetyl chloride. Themixture was stirred at room temperature for 30 minutes, filtered and thefiltrate was added dropwise to 500 ml of ether. The solid that separatedwas filtered and dried in a vacuum desiccator.

Example 20 4-Dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 20, in 10 ml of 0.1N hydrochloric acid in methanol cooled in anice bath was added 0.5 ml of n-butyl nitrite. The solution was stirredat ice bath temperature for 30 minutes and then poured into 250 ml ofether. The solid that separated was filtered, washed with ether anddried in a vacuum desiccator.

Example 21 7-Azido-4-dedimethylamino-6-deoxy-5-hydroxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, 10 ml of 0.1 N methanolic hydrogenchloride was added 0.33 mmole of sodium azide. The mixture was stirredat room temperature for 1.5 hours. The reaction mixture was then pouredinto 200 ml of ether. The solid that separated was filtered and dried ina vacuum desiccator.

Example 227-Amino-8-chloro-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxy-5-hydroxytetracyclinesulfate, from example 21, was dissolved in 10 ml of concentratedsulfuric acid (previously saturated with hydrogen chloride) at 0° C. Themixture was stirred at ice bath temperature for 1.5 hours and thenslowly added dropwise to 500 ml of cold ether. The solid that separatedwas filtered, washed with ether and dried in a vacuum desiccator.

Example 234-Dedimethylamino-6-deoxy-5-hydroxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, in 15 ml of water was added to asolution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water.The mixture was stirred at room temperature for one hour. The solid thatseparated was filtered and dried in a vacuum desiccator.

Example 247-Dimethylamino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of a 40% aqueous formaldehyde solution and 100 mg of a 10%palladium on carbon catalyst. The mixture is reduced with hydrogen atatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 78 mg.

Example 25 7-Diethylamino-4-dedimethylamino-5-hydroxytetracyclinesulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of acetaldehyde and 100 mg of a 10% palladium on carbon catalyst.The mixture is reduced with hydrogen at atmospheric pressure at roomtemperature for 20 minutes. The catalyst was filtered and filtrate wasevaporated to dryness under reduced pressure. The residue is dissolvedin 5 ml of methanol and this solution was added to 100 ml of ether. Theproduct that separated was filtered and dried.

Example 26 4-Dedimethylamino-6-deoxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g. of 7-amino-4-dedimethylamino-6-deoxytetracyclinesulfate, from example 17, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 27 7-Azido-4-dedimethylamino-6-deoxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33mmole of sodium azide. The mixture was stirred at room temperature for1.5 hours. The reaction mixture was then poured into 200 ml of ether.The solid that separated was filtered and dried in a vacuum desiccator.

Example 28 7-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclinesulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracycline sulfatewas dissolved in 10 ml of concentrated sulfuric acid (previouslysaturated with hydrogen chloride) at 0° C. The mixture was stirred atice bath temperature for 1.5 hours and then slowly added dropwise to 500ml of cold ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 294-Dedimethylamino-6-deoxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, in 15 ml of water was added to a solution of 1.15 mmole ofpotassium ethyl xanthate in 15 ml of water. The mixture was stirred atroom temperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 30 7-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound, from example 26, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is reduced withhydrogen at atmospheric pressure and room temperature for 20 minutes.The catalyst was filtered and the filtrate was evaporated to drynessunder reduced pressure. The residue is dissolved in 5 ml of methanol andthis solution was added to 100 ml of ether. The product that separatedwas filtered and dried.

Example 319-Acetamido-8-chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyltetracycline

To well stirred cold solution of 500 mg of9-amino-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethyl aminotetracycline sulfate, from example 6, in 2.0 ml of1,3-dimethyl-2-imidazolidinone was added 500 mg of sodium bicarbonatefollowed by 0.21 ml. of acetyl chloride. The mixture was stirred at roomtemperature for 30 minutes, filtered and the filtrate was added dropwiseto 500 ml of ether. The solid that separated was filtered and dried in avacuum desiccator.

Example 328-Chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyl-9-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethylamino-9-diazoniumtetracycline hydrochloride in 15 ml of water was addedto a solution of 1.15 mmole of potassium ethyl xanthate in 15 ml ofwater. The mixture was stirred at room temperature for one hour. Thesolid that separated was filtered and dried in a vacuum desiccator.

Example 338-Chloro-9-dimethylamino-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethytetracyclinesulfate

To a solution of 100 mg. of the 9-amino compound, from example 6, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of acetaldehyde and 100 mg of a 10% palladium oncarbon catalyst. The mixture is reduced with hydrogen at atmosphericpressure and room temperature for 20 minutes. The catalyst was filteredand the filtrate was evaporated to dryness under reduced pressure. Theresidue is dissolved in 5 ml of methanol and this solution was added to100 ml of ether. The product that separated was filtered and dried.

Example 34 N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxytetracycline

An aqueous solution of 58 mg (37%) formaldehyde (0.72 mmol) was added toa solution of 203 mg (0.49 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 5.0 ml ethyleneglycol dimethyl ether. The mixture was stirred at room temperature for0.5 hours. 56 mg (0.56 mmol) of 1-methylpiperazine was then added andthe resulting mixture was stirred overnight and refluxed for 20 minutes.The mixture was then cooled and a solid product was collected byfiltration. The solid product was then washed with the solvent and driedby vacuum filtration.

Example 35N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

An aqueous solution of 49 mg 37% formaldehyde (0.60 mmol) was added to asolution of 146 mg (0.30 mmol) of4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline in 5.0ml ethylene glycol dimethyl ether. The mixture was stirred at roomtemperature for 0.5 hours. 60 mg (0.60 mmol) of 1-methylpiperazine wasthen added and the resulting mixture was stirred overnight and refluxedfor 20 minutes. The mixture was then cooled and a solid product wascollected by filtration. The solid product was then washed with thesolvent and dried by vacuum filtration.

Example 364-Dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline.

1.54 g (7.2 mmol) of hexanoic anhydride and 150 mg of 10% Pd/C catalystwere added to 300 mg (0.72 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 6.0 ml of1,4-dioxane and 6.0 ml of methanol. The mixture was hydrogenatedovernight at room temperature. The catalyst was removed by filtrationand the filtrate was concentrated under reduced pressure. The residuewas dissolved in 7 ml of ethyl acetate and trituated with 50 ml ofhexane to produce a solid product. The solid product was filtered anddried by vacuum filtration.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

Example 37 Phototoxicity Determination

BALB/c 3T3 (CCL-163) cells were obtained from ATCC and cultured inantibiotic-free Dulbecco's Minimum Essential Medium (4.5 g/lglucose)(DMEM) supplemented with L-glutamine (4 mM) and 10% newborn calfserum. The working cell bank was prepared and found to be free ofmycoplasma. Streptomycin sulfate (100 μg/ml) and penicillin (100 IU/ml)were added to the medium after the cells were treated with test articlein 96-well plates.

Serial dilutions of the tetracycline derivatives were prepared in DMSOat concentrations 100× to final testing concentration. The CMT dilutionsin DMSO were then diluted in Hanks' Balanced Salt Solution (HBSS) forapplication to the cells. The final DMSO concentration was 1% in treatedand control cultures. For the dose range finding assay, 8 serialdilutions covered a range of 100 to 0.03 mg/ml in half log steps whilethe definitive assays used 6 to 8 doses prepared in quartet log steps,centered on the expected 50% toxicity point. In many cases, the doserange for treatment without UV light was different from the dose rangeselected with UV light. One hundred μg/ml is the highest doserecommended to prevent false negative results from UV absorption by thedosing solutions.

Controls: Each assay included both negative (solvent) and positivecontrols. Twelve wells of negative control cultures were used on each96-well plate. Chlorpromazine (Sigma) was used as the positive controland was prepared and dosed like the test tetracycline derivatives.

Solar Simulator: A Dermalight SOL 3 solar simulator, equipped with a UVAH1 filter (320-400 nm), was adjusted to the appropriate height.Measurement of energy through the lid of a 96-well microtiter plate wascarried out using a calibrated UV radiometer UVA sensor. Simulatorheight was adjusted to deliver 1.7±0.1 m/Wcm² of UVA energy (resultingdose was 1 J/cm² per 10 min.)

Phototoxicity Assay: Duplicate plates were prepared for each testmaterial by seeding 10⁴ 3T3 cells per well in μl of complete medium 24hours before treatment. Prior to treatment, the medium was removed, andthe cells washed once with 125 μl prewarmed HBSS. Fifty μl of prewarmedHBSS were added to each well. Fifty μl of test article dilutions wereadded to the appropriate wells and the plates returned to the incubatorfor approximately one hour. Following the 1 hr incubation, the platesdesignated for the photoirritation assay were exposed (with the lid on)to 1.7±0.1 mW/cm² UVA light for 50±2 minutes at room temperatureresulting in an irradiation dose of 5 J/cm2. Duplicate plates designatedfor the cytotoxicity assay were kept in the dark room temperature for50±2 minutes. After the 50 minute exposure period the test articledilutions were decanted from the plates and the cells washed once with125 μl HBSS. One hundred μl of medium were added to all wells and thecells incubated as above for 24±1 hours.

After 24 hours of incubation, the medium was decanted and 100 μl of theNeutral Red containing media added to each well. The plates werereturned to the incubator and incubated for approximately 3 hours. After3 hours, the medium was decanted and each well rinsed once with 250 μlof HBSS. The plates were blotted to remove the HBSS and 100 μl ofNeutral Red Solvent were added to each well. After a minimum of 20minutes of incubation at room temperature (with shaking), the absorbanceat 550 nm was measured with a plate reader, using the mean of the blankouter wells as the reference. Relative survival was obtained bycomparing the amount of neutral red taken by test article and positivecontrol treated groups to the neutral red taken up by the negative groupon the same plate. IC₅₀ values for both the UVA exposed and non-exposedgroups were determined whenever possible. One dose range finding and atleast two definitive trails were performed on each tetracyclinederivative and control compound.

Determination of Phototoxicity: Phototoxicity of the tetracyclinederivatives can be measured by its photoirritancy factor (PIF). The PIFwas determined by comparing the IC₅₀ without UVA [IC₅₀(−UVA)] with theIC₅₀ with UVA [IC₅₀(+UVA)]:

${PIF} = \frac{{IC}_{50}\left( {- {UVA}} \right)}{{IC}_{50}\left( {+ {UVA}} \right)}$

If both IC₅₀ values can be determined, the cut off value of the factorto discriminate between phototoxicants and non-phototoxicants is afactor of 5. A factor greater than 5 is indicative of phototoxicpotential of the test material.

If IC₅₀ (+UVA) can be determined but IC₅(−UVA) cannot, the PIF cannot becalculated, although the compound tested may have some level ofphototoxic potential. In this case, a “>PIF” can be calculated and thehighest testable dose (−UVA) will be used for calculation of the “>PIF.”

${> {PIF}} = \frac{{maximum}\mspace{14mu}{dose}\mspace{11mu}\left( {- {UVA}} \right)}{{IC}_{50}\left( {+ {UVA}} \right)}$

If both, IC₅₀(−UVA) and IC₅₀(+UVA) cannot be calculated because thechemical does not show cytotoxicty (50% reduction in viability) up tothe highest dose tested, this would indicate a lack of phototoxicpotential.

INDEX OF STRUCTURES

1. A method for treating a mammal suffering from a condition thatbenefits from a non-antimicrobial dose of a tetracycline compound, saidcondition being characterized by excessive collagen destruction,excessive MMP enzyme activity, excessive TNF activity, excessive nitricoxide activity, excessive IL-1 activity, excessive elastase activity,excessive loss of bone density, excessive protein degradation, excessivemuscle wasting, excessive glycosylation of collagen, excessive COX-2activity, insufficient bone protein synthesis, insufficientinterleukin-10 production or excessive phospholipase A₂ activity, themethod comprising administering to the mammal an effective amount of a4-de(dimethylamino) doxycycline compound having structure L, M, N, or O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen palmitamide (COL-803) hydrogenhydrogen nitro (COL-804) hydrogen hydrogen amino (COL-805)

wherein said condition is abdominal aortic aneurysm, ulceration of thecornea, periodontal disease, diabetes, diabetes mellitus, scleroderma,progeria, graft versus host disease, disease of depressed bone marrowfunction, thrombocytopenia, prosthetic joint loosening,spondyloarthropathies, osteoporosis, Paget's disease, systemic lupuserythematosis, inflammatory bowel disease, arthritis, osteoarthritis,rheumatoid arthritis, pancreatitis, nephritis, glomerulonephritis,sepsis, septic shock, lipopolysaccharide endotoxin shock, multisystemorgan failure, psoriasis, ARDS, cystic fibrosis, emphysema, acute lunginjury resulting from inhalation of toxicants, chronic renal failure,acute renal failure, nephritis or glomerulonephritis.
 2. A method fortreating a mammal suffering from a neurological or neurodegenerativecondition that benefits from a non-antimicrobial dose of a tetracyclinecompound, the method comprising administering to the mammal an effectiveamount of a 4-de(dimethylamino)doxycycline compound having structure L,M, N, or O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen palmitamide (COl-803) hydrogenhydrogen nitro (COL-804) hydrogen hydrogen amino (COL-805)

wherein the neurological or neurodegenerative condition is Alzheimer'sdisease, Guillain-Barré Syndrome, Krabbe's disease,adrenoleukodystrophy, Parkinson's disease, Huntington's disease,multiple sclerosis, amyotrophic lateral sclerosis or an encephalopathy.3. A method according to claim 2, wherein said encephalopathy is aspongiform encephalopathy.